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Intramural Papers of the Month

By Robin Arnette
January 2009

Damaged Single-Strand DNA Formed at Double-Strand Breaks and Uncapped Telomeres

In an article published in PLoS Genetics, NIEHS scientists reported that many kilobases of single-strand DNA created in the vicinity of double-strand breaks or at uncapped telomeres of the chromosomes of the budding yeast Saccharomyces cerevisiae, can be restored to viable double-strand form even if it contains multiple damages. In the process of such restoration the damages were turned into mutations by error-prone translesion DNA polymerase zeta.

The researchers wanted to investigate the molecular mechanisms that could provide simultaneous mutations long sought as a source of high fitness alleles in evolution and cancer. Team members constructed yeast strains using PCR-based gene disruption and genome modification by oligonucleotides. They then assessed the strains for mutagenesis associated with double-strand break repair and uncapped telomere arrest.

This work establishes a simple molecular mechanism for simultaneous generation of multiple mutations spanning over several kilobases without severe mutation load in the rest of the genome and puts forward questions about the share of damaged single-strand DNA in various kinds of environmental, occupational and drug-related mutagenesis. It also suggests a tool for genome-wide detection of transient long single-strand DNA regions formed during DNA replication double-strand break repair and telomere metabolism.

Citation: Yang Y, Sterling J, Storici F, Resnick MA, Gordenin DA. (http://www.ncbi.nlm.nih.gov/pubmed/19023402?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2008. Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae. PLoS Genetics 4(11):e1000264.

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Rhox13 Is a Novel Rhox Gene in Rats and Mice

Found on the X chromosome of rodents, the reproductive homeobox (Rhox) gene cluster contains over 30 genes encoding transcription factors involved in development of various reproductive organs. However, recent studies performed by researchers at NIEHS have discovered a novel Rhox gene that is also located on chromosome X, but does not appear to be expressed in Sertoli cells or extraembryonic tissues like other Rhox genes. Called Rhox13, the gene may regulate a subset of genes in the prenatal ovary as well as the prenatal and postnatal testis.

The investigators knew that the previously characterized reproductive Rhox genes overlapped each other in terms of expression, so they set out to fully characterize this gene. The team used several techniques including northern blotting, RT-PCR, western blotting, immunohistochemistry and expression of recombinant RHOX13.

Rhox13 is similar to other Rhox genes in that it has a homeobox domain and lacks a paired domain, but its expression pattern is uniquely germ cell-specific. Rhox13 transcripts are first found in the testis and ovary at 13.5 days post coitum (dpc). Rhox13 transcription in the testes continues through adulthood, while in the ovary, transcription stops by three days post partum (dpp). The RHOX13 protein is expressed in a subset of germ cells, namely those spermatogonia and oogonia that have differentiated to enter meiosis. Future studies will be aimed at revealing the role of Rhox13 in differentiation of germ cells in the testis and ovary.

Citation: Geyer CB, Eddy EM. (http://www.ncbi.nlm.nih.gov/pubmed/18675325?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2008. Identification and characterization of Rhox13, a novel X-linked mouse homeobox gene. Gene 423(2):194-200.

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The DSS1 Protein Requires the R3IM Motif for Proteasome Interaction

A recent study determined that human Deleted in Split hand/Split foot 1 (HsDSS1) formed a complex with the proteasome via a specific subunit, which then bound with the ubiquitin-proteasome system (UPS), thus regulating ubiquitin-mediated protein degradation. Specifically, the amino-terminus of HsDSS1 contained an RPN3/S3-interacting motif (R3IM) that interacted with RPN3/S3, and this interaction had the capacity to not only control UPS, but also alter the steady state levels of important ubiquinated substrates such as the tumor suppressor protein, p53. The work was performed collaboratively by a group of scientists from NIEHS and Alpha-Gamma Technologies Inc.

The investigators used several research methods to uncover DSS1's role in eukaryotic organisms, but one experiment, using siRNA to knockdown endogenous levels of HsDSS1, resulted in an increase in p53. This study was the first to demonstrate this action. The team uncovered several other findings from the data.

  • An Asp/Glu-rich region of the R3IM in HsDSS1 regulated the proteasome interaction and degradation of ubiquitin-conjugated substrates.
  • The interaction between HsDSS1 and RPN3/S3 was highly conserved, from nematodes to humans.
  • The HsDSS1/RPN3/S3 proteasome complex was required for p53 targeting.
  • The PCI/PINT domain, located within the RPN3/S3, was needed for binding to HsDSS1 and the proteasome.

The research has implications in many disease states such as cancer, aging, inflammation-related and neurodegenerative disorders.

Citation: Wei SJ, Williams JG, Dang H, Darden TA, Betz BL, Humble MM, Change FM, Trempus CS, Johnson K, Cannon RE, Tennant RW. (http://www.ncbi.nlm.nih.gov/pubmed/18775730?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2008. Identification of a specific motif of the DSS1 protein required for proteasome interaction and p53 protein degradation. J Mol Biol 383(3):693-712.

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The Nrf2 Pathway Protects Against RSV Disease in Rodents

Researchers from NIEHS, Johns Hopkins University School of Medicine, the INFANT Foundation in Argentina and the Tohoku University Graduate School of Medicine in Japan determined that the Nrf2-antioxidant response element (ARE) pathway played a key role in protecting murine airways against respiratory syncytial virus (RSV) disease and oxidative stress. The finding is the first to demonstrate the importance of Nrf2-mediated antioxidant mechanisms in host viral infection and provides a possible means for therapeutic intervention.

RSV, the leading cause of severe lower airway disease in infants, young children and immune compromised adults, is characterized by bronchiolitis and respiratory failure in highly susceptible individuals. Because of these health implications, the investigators wanted to understand the pathogenesis of the infection. The team had two major objectives: (1) To determine the functional role of Nrf2 in RSV disease, and (2) whether the induction of Nrf2 and antioxidant enzymes would lessen the oxidant airway injury by RSV.

To address the first goal, the team infected Nrf2-deficient mice (Nrf2-/-) and wild-type mice (Nrf2+/+) with RSV or vehicle. Nrf2-/-mice exhibited augmented bronchopulmonary inflammation and injury and suppressed antioxidant induction compared to Nrf2+/+mice. For the second aim, Nrf2-/- and Nrf2+/+ mice were orally treated with sulphoraphane, a naturally occurring antioxidant and Nrf2-ARE inducer, prior to infecting with RSV. Sulphoraphane pre-treatment dramatically limited lung RSV replication and virus-induced inflammation in Nrf2+/+ but not in Nrf2-/- mice. This research suggests that targeting oxidative stress may limit RSV infectivity and improve human respiratory health.

Citation: Cho HY, Imani F, Miller-Degraff L, Walters D, Melendi GA, Yamamoto M, Polack FP, Kleeberger SR. (http://www.ncbi.nlm.nih.gov/pubmed/18931336?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum) Exit NIEHS 2008. Antiviral activity of Nrf2 in a murine model of respiratory syncytial virus (RSV) disease. Am J Respir Crit Care Med [Epub ahead of print].



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